Multi-part differential analyzing apparatus utilizing light scatter techniques

Abstract

Disclosed is a flow through particle analyzing cell differentiating apparatus for optical and electronic measurements on a stream of particles in which a hydrodynamically focoussed stream is passed into and through a point focussed beam of radiated energy whereby the beam is scattered by the stream to impinge upon light responsive signal operating members disposed adjacent the cell at angular positions relative to the beam axis of from about 10° to 70°.

What we claim is: 
     
       1. Apparatus for selectively differentiating at least one white blood cell subpopulation in a blood sample, whereby differentiation is based upon individual cell light scatter, said apparatus comprising: a cytometric flow cell including inlet and outlet means for the ingress and egress of a blood sample in liquid suspension;   introducing means for introducing a blood sample including white cells into said flow cell inlet to cause said white cells to flow through said flow cell;   a beam of light arranged such that the axis of its light rays pass through said flow cell at right angles to said flow of white cells;   extinguishing means, axially aligned with said axis of light rays, for extinguishing the light rays which are at a low angle to said beam axis which are scattered by the white cells in response to the impingement of light thereon as they pass through said beam;   light collection means responsive to the light scattered by said cells in a median angle (MALS) greater than 10°, but less than 70°, relative to said axis after passing through said beam for producing median angle output signal data, eosinophil subpopulation data being generated by and derived from the scattered rays in the (MALS) range of 20° to 70°;   utilizing means for utilizing said output signal data from said light collection means for utilizing the portion of said (MALS) in the range of 20° to 70° to differentiate the eosinophil subpopulation; and   said apparatus is constructed to operate to differentiate white blood cells, especially including eosinophil blood cells, as a result of the inherent, natural characteristics of the white blood cells and without treatment of any kind for specifically altering the inherent, natural characteristics of the white blood cells for differentiating eosinophils.   
     
     
       2. Apparatus in accordance with claim 1 wherein said means for extinguishing said small angle light rays comprises a horizontally disposed light interrupting means is positioned to intercept and block all but the scattered light rays in the range of 20° to 60°. 
     
     
       3. Apparatus in accordance with claim 1 wherein said extinguishing means for selectively occluding said light rays includes angularly disposed apertures therein permitting the passage of said light rays at prescribed angles prior to impingement upon said light collection means. 
     
     
       4. Apparatus in accordance with claim 3 wherein at least one of said apertures is located on the axis of said light rays leaving said flow cell. 
     
     
       5. Apparatus in accordance with claim 1 wherein said light collection means includes means disposed on said light beam axis responsive to axial light loss. 
     
     
       6. Apparatus in accordance with claim 1 in which said light collection means comprises individual members disposed at 45° relative the axis of said light beam. 
     
     
       7. Apparatus in accordance with claim 1 wherein said flow cell is circular in cross section. 
     
     
       8. Apparatus in accordance with claim 1 wherein said flow cell is square in cross section intermediate said inlet and outlet. 
     
     
       9. Apparatus in accordance with claim 1 wherein said light collection means is disposed adjacent to at least three sides of said flow cell. 
     
     
       10. Apparatus in accordance with claim 1 wherein said light collection means is constructed and arranged with respect to construction and arrangement of said light extinguishing means to collect and respond to light scattered by said sample in the angular range of 20° to 70° relative to the axis of the light leaving said flow cell aperture. 
     
     
       11. Apparatus in accordance with claim 1 further including first electronic circuit means for generating: a first signal of Coulter DC volume; said first electronic circuit means employs said output signal from said light collection means (MALS) in the range of 10°-70° for generating a second signal of log of median angle light scatter (log of MALS); and a first output is developed from said first and second signals as rotated light scatter (RLS). 
     
     
       12. Apparatus in accordance with claim 11 including second electronic circuit means for generating: a third signal of Coulter RF, and generating a second output of Coulter opacity, which is produced by and results from said first signal of Coulter DC volume and said third signal of Coulter RF. 
     
     
       13. Apparatus according to claim 12 constructed and arranged such that: said first output of RLS yields data specific to the white cell subpopulation of neutrophils, separate from the eosinophils, and also a group of three subpopulations of lymphocytes, monocytes, and basophils; said first signal of Coulter DC volume yields data which separates the monocytes from said group of three subpopulations; and said second output of Coulter opacity yields data which separates the lymphocytes and basophils from said group of three subpopulations; whereby said apparatus differentiates the five white cell subpopulations of: eosinophils, neutrophils, monocytes, lymphocytes, and basophils. 
     
     
       14. Apparatus in accordance with claim 1 including first electronic circuit means for generating: a first signal of Coulter RF; and generating a second signal which employs said output signal from said light collection means (MALS) for producing log of median angle light scatter (log of MALS); and second electronic circuit means for comparing said first signal of Coulter RF and said second signal of log of MALS whereby five subpopulations of white blood cells can be differentiated. 
     
     
       15. A method for differentiating at least one white blood cell subpopulation in a blood sample, whereby differentiation is based upon individual cell light scatter, said method comprising the steps of: passing a beam of light along an axis and orthogonally through a fluid passageway while simultaneously flowing said blood sample through said passageway; extinguishing the rays of light which are at a low angel to said axis which are scattered by the white cells in response to impingement of light thereon as they pass through said beam of light; collecting the light scattered by said cells in a median angle (MALS) greater than 10°, but less than 70°, relative to said axis after passing through said beam of light; sensing the rays of light scattered in said median angle by each cell of said blood sample for producing median angle output signal data; said step of sensing providing signal data which is a result of the inherent, natural light scatter characteristics of the white blood cells without treatment of any kind for specifically altering the inherent, natural characteristics of said white blood cells for differentiating eosinophils, eosinophil data being contributed by the portion of (MALS) from 20° to 70°; and utilizing said portion of said median angle light scatter signal data to differentiate the eosinophil subpopulation. 
     
     
       16. The method of claim 15 in which said step of flowing includes the steps of providing said fluid passageway within a Coulter volume fluid flow chamber, hydrodynamically focussing said blood sample as it is flowing through said chamber, generating Coulter volume DC signal information (DC), comparing said DC with signal information obtained from the light scatter sensing, whereby said processing produces data which differentiates five distinct white cell subpopulations, namely: eosinophils, neutrophils, monocytes, lymphocytes and basophils. 
     
     
       17. The method of claim 15 including the additional processing step of developing a log of median angle light scatter signal information (log of MALS) from said MALS for classifying said white blood cells into three distinct groups, namely: eosinophils, neutrophils, and lymphocytes, monocytes and basophils. 
     
     
       18. The method of claim 17 including the steps of generating Coulter volume DC signal information (DC) and dividing the log of MALS by said DC for generating rotated light scatter signal information (RLS). 
     
     
       19. The method of claim 18 including the step of comparing said RLS with said DC, whereby said processing produces data for differentiating at least five white blood cell subpopulations, namely: eosinophils, neutrophils, lymphocytes, monocytes and basophils. 
     
     
       20. The method of claim 18 wherein RLS is analyzed in a histogram providing signal information to yield three separate populations: eosinophils, neutrophils, and a combination population including lymphocytes, monocytes and basophils. 
     
     
       21. The method of claim 20 including the steps of gating on the RLS data to produce a DC histogram separated so as to provide up to at least three separate populations: monocytes, a mixture of lymphocytes and basophils, and low volume lymphocytes. 
     
     
       22. The method of claim 21 further including the steps of generating a Coulter RF signal value information (RF), dividing said RF by said DC to produce an opacity signal, and including the further step of gating on the DC signal information to produce an opacity histogram separated so as to provide up to at least three separate populations: basophils, lymphocytes, and low opacity lymphocytes. 
     
     
       23. The method of claim 20 including the steps of gating on the RLS signal information to produce a DC histogram containing at least a population of normal neutrophils and possibly including a second population of other cell types. 
     
     
       24. The method of claim 23 further including the steps of generating a Coulter RF signal value information (RF), dividing said RF by said DC to produce an opacity signal, and including the step of gating on the DC signal information to produce an opacity histogram of said other cell types which are classified as damaged neutrophils and lymphocytes. 
     
     
       25. The method of claim 17 including the steps of generating Coulter volume DC signal information (DC) and comparing said log of MALS with said DC, whereby said processing produces data for differentiating at least five white blood cell subpopulations, namely: eosinophils, neutrophils, lymphocytes, monocytes and basophils. 
     
     
       26. The method of claim 17 including the steps of generating a Coulter RF signal value information (RF) and comparing said log of MALS and said RF, whereby said processing produces data for differentiating five subpopulations of white blood cells, namely: eosinophils, neutrophils, monocytes, lymphocytes and basophils. 
     
     
       27. The method of claim 17 including the additional steps of obtaining narrow angle light scatter signal information (NALS) and thereafter comparing said log of MALS with said NALS, whereby said processing produces data for differentiating four white blood cell subpopulations, namely: lymphocytes, monocytes, neutrophils and eosinophils. 
     
     
       28. The method of claim 17 including the additional sensing steps of obtaining axial light loss signal information (ALL) and comparing said log of MALS with said ALL, whereby said processing produces data for differentiating five white blood cell subpopulations, namely: eosinophils, neutrophils, monocytes, lymphocytes and basophils. 
     
     
       29. The method of claim 15 including the additional steps of obtaining 15° light scatter signal information (15° LS) and axial light loss signal information (ALL), and comparing said 15° LS with said ALL, whereby said processing produces data for differentiating five subpopulations of white blood cells, namely: eosinophils, neutrophils, monocytes, lymphocytes and basophils. 
     
     
       30. The method of claim 15 including the additional steps of obtaining axial light loss signal information (ALL), generating Coulter volume DC signal information (DC), and comparing said ALL with said DC for producing data for differentiating three white blood cell subpopulations, namely: lymphocytes, basophils and eosinophils, monocytes and neutrophils. 
     
     
       31. The method of claim 15 including the steps of applying simultaneously to said flow cell passageway a Coulter volume DC signal (DC) and a Coulter RF signal (RF), and comparing said DC to said RF, whereby said processing provides data for differentiating an additional four white blood cell subpopulations, namely: neutrophils, monocytes, lymphocytes and basophils. 
     
     
       32. The method of claim 31 including the steps of electronically dividing said RF by said DC to produce an opacity signal which is independent of blood cell volume and is related to blood cell internal conductivity, and comparing said opacity signal to said DC, whereby said processing provides data for enhancing the differentiating of said four white blood cell subpopulations. 
     
     
       33. The method of claim 15 including the step of conditioning said white blood cells such that they approximate native state as a result of the inherent, natural characteristics of the whole blood cell and not as a result of a treatment for specifically altering the characteristics of said whole blood cells for differentiating eosinophils while said cells are flowing through said passageway. 
     
     
       34. A method for differentiating at least one white blood cell subpopulation in a blood sample, whereby differentiation is based upon individual cell light scatter, said method comprising the steps of: passing rays of light along an axis and orthogonally through a fluid passageway while simultaneously flowing said blood sample through said passageway; extinguishing the rays of light which are at a low angle to said axis which are scattered by the white cells in response to impingement of light therein as they pass through said beam of light; collecting the light scattered by said cells in a median angle (MALS) greater than 10°, but less than 70°, relative to said axis after passing through said beam of light; sensing the rays of light scattered in said median angle for producing median angle output signal data by each cell of said blood sample; eosinophil subpopulation data being generated by and derived from the scattered rays in the (MALS) range of 20° to 70°; said step of sensing providing signal data as a result of the inherent, natural light scatter characteristics of the white blood cells without treatment of any kind for specifically altering the inherent, natural characteristics of said white blood cells for differentiating white blood cell subpopulations; and processing said output signal data to produce data for utilizing the portion of said (MALS) in the range of 20° to 70° to differentiate at least three subpopulations, namely eosinophils, neutrophils, and lymphocytes, monocytes and basophils. 
     
     
       35. A method for differentiating at least one white blood cell subpopulation in a blood sample, whereby differentiation is based upon individual cell light scatter, said method comprising the steps of: passing rays of light along an axis and orthogonally through a fluid passageway while simultaneously flowing said blood sample through said passageway; extinguishing the rays of light which are at a low angle to said axis which are scattered by the white cells in response to impingement of light thereon as they pass through said beam of light; collecting the light scattered by said cells in a median angle (MALS) greater than 10°, but less than 70°, relative to said axis after passing through said beam of light; sensing the rays of light scattered in said median angle by each cell of said blood sample for producing median angle output signal data; said step of sensing provides signal data as a result of the inherent, natural light scatter characteristics of the white blood cells without treatment of any kind for specifically altering the inherent, natural characteristics of said white blood cells for differentiating white blood cell subpopulations and is within a collection angle of from 10° to 70° relative to said axis; processing electronically said output signal data to produce white blood cell differentiating data; wherein said step of sensing is operative with the untreated white blood cells and within a collection angle of from 10° to 70° relative to said axis; eosinophil subpopulation data being generated by and derived from the scattered rays in the (MALS) range of 20° to 70°; and including the additional steps of obtaining axial light loss signal information (ALL), generating Coulter volume DC signal information (DC), and comparing said ALL with said DC for producing data for differentiating three white blood cell subpopulations, namely: lymphocytes, basophils and eosinophils, monocytes and neutrophils. 
     
     
       36. The method of claim 35 including the further step of dividing said ALL by said DC, whereby said processing produces data for differentiating at least three white blood cell subpopulations, namely: lymphocytes, basophils and eosinophils, monocytes and neutrophils. 
     
     
       37. A method for differentiating at least one white blood cell subpopulation in a blood sample, whereby differentiation is based upon individual cell light scatter, said method comprising the steps of: passing rays of light along an axis and orthogonally through a fluid passageway while simultaneously flowing said blood sample through said passageway; extinguishing the rays of light which are at a low angle to said axis which are scattered by the white cells in response to impingement of light thereon as they pass through said beam of light; collecting the light scattered by said cells in a median angle (MALS) greater than 10°, but less than 70°, relative to said axis after passing through said beam of light; sensing the rays of light scattered in said median angle by each cell of said blood sample for producing median angle output signal data; said step of sensing providing signal data as a result of the inherent, natural light scatter characteristics of the white blood cells without treatment of any kind for specifically altering the inherent, natural characteristics of said white blood cells for differentiating white blood cell subpopulations; processing electronically the said output signal data to produce white blood cell differentiating data; wherein said step of sensing is operative with the untreated white blood cells and within a collection angle of from 10° to 70° relative to said axis; and including the steps of applying a DC and an RF current simultaneously to said flow cell passageway aperture producing a Coulter volume DC signal (DC) and a Coulter RF signal (RF), and comparing said DC to said RF, whereby said processing provides data for differentiating at least four white blood cell subpopulations, namely: neutrophils, monocytes, lymphocytes, and basophils. 
     
     
       38. A method for differentiating at least one white blood cell subpopulation in a blood sample, whereby differentiation is based upon individual cell light scatter, said method comprising the steps of: passing rays of light along an axis and orthogonally through a fluid passageway including a Coulter aperture while simultaneously flowing said blood sample through said passageway; sensing some of the rays of light scattered by each cell of said blood sample; processing electronically the results of said sensing step to produce white blood cell differentiating data; wherein said step of sensing provides median angle light scatter (MALS) signal data throughout a range of approximately 10°-70° and wherein said processing step develops a log of median angle light scatter signal (log of MALS); including the steps of applying a DC current simultaneously to said flow cell passageway aperture producing a Coulter volume DC signal (DC), dividing the log of MALS by said DC for generating rotated light scatter signal information (RLS) and comparing said DC with said RLS, whereby said processing provides data for differentiating and identifying at least four white blood cell subpopulations, namely: lymphocytes and basophils, monocytes, neutrophils, and eosinophils. 
     
     
       39. A method for differentiating at least one white blood cell subpopulation in a blood sample, whereby differentiation is based upon individual cell light scatter, said method comprising the steps of: passing rays of light along an axis and orthogonally through a fluid passageway including a Coulter aperture while simultaneously flowing said blood sample through said passageway, sensing some of the rays of light scattered by each cell of said blood sample; processing electronically the results of said sensing step to produce white blood cell differentiating data, wherein said step of sensing provided median angle light scatter (MALS) signal data throughout a range of approximately 10°-70° and said processing step develops a log of median angle light scatter signal (log of MALS), applying a DC and an RF current simultaneously to said flow cell passageway aperture for producing a Coulter volume DC signal (DC), dividing the log of MALS by said DC for generating rotated light scatter signal information (RLS), and comparing said DC with said RLS, whereby said processing provides data for differentiating and identifying at least four white blood cell subpopulations, namely: lymphocytes and basophils, monocytes, neutrophils, and eosinophils, dividing said RF by said DC for generating opacity signal information (OP), and comparing said DC with said OP while gating on said RLS, whereby said processing provides data for further differentiating and identifying one of said four white blood cell subpopulations into at least two white blood cell subpopulations, namely: lymphocytes and basophils. 
     
     
       40. A method for differentiating at least one white blood cell subpopulation in a blood sample, whereby differentiation is based upon individual cell light scatter, said method comprising the steps of: passing rays of light along an axis and orthogonally through a fluid passageway including a Coulter aperture while simultaneously flowing said blood sample through said passageway; sensing some of the rays of light scattered by each cell of said blood sample; processing electronically the results of said sensing step to produce white blood cell differentiating data; wherein said step of sensing provides median angle light scatter (MALS) signal data throughout a range of approximately 10°-70° and said processing step develops a log of median angle light scatter signal (log of MALS); applying a DC and an RF current simultaneously to said flow cell passageway aperture for producing a Coulter volume DC signal (DC), dividing the log of MALS by said DC for generating rotated light scatter signal information (RLS), dividing said RF by said DC for generating opacity signal information (OP), and comparing said DC with said RLS and said OP, whereby said processing provides data for differentiating and identifying at least five white blood cell populations, namely: lymphocytes, basophils, monocytes, neutrophils, and eosinophils.